Targeting of essential mycobacterial replication enzyme DnaG primase revealed Mitoxantrone and Vapreotide as novel mycobacterial growth inhibitors.

Tuberculosis (TB) is the second leading cause of mortality after COVID-19, with a global death toll of 1.6 million in 2021. The escalating situation of drug-resistant forms of TB has threatened the current TB management strategies. New therapeutics with novel mechanisms of action are urgently required to address the current global TB crisis. The essential mycobacterial primase DnaG with no structural homology to homo sapiens presents itself as a good candidate for drug targeting. In the present study, Mitoxantrone and Vapreotide, two FDA-approved drugs, were identified as potential anti-mycobacterial agents. Both Mitoxantrone and Vapreotide exhibit a strong Minimum Inhibitory Concentration (MIC) of ≤25µg/ml against both the virulent (M.tb-H37Rv) and avirulent (M.tb-H37Ra) strains of M.tb. Extending the validations further revealed the inhibitory potential drugs in ex vivo conditions. Leveraging the computational high-throughput multi-level docking procedures from the pool of ~2700 FDA-approved compounds, Mitoxantrone and Vapreotide were screened out as potential inhibitors of DnaG. Extensive 200 ns long all-atoms molecular dynamic simulation of DnaGDrugs complexes revealed that both drugs bind strongly and stabilize the DnaG during simulations. Reduced solvent exposure and confined motions of the active centre of DnaG upon complexation with drugs indicated that both drugs led to the closure of the active site of DnaG. From this study's findings, we propose Mitoxantrone and Vapreotide as potential anti-mycobacterial agents, with their novel mechanism of action against mycobacterial DnaG.

Unravelling the potential of Triflusal as an anti-TB repurposed drug by targeting replication protein DciA.

The increasing prevalence of drug-resistant Tuberculosis (TB) is imposing extreme difficulties in controlling the TB infection rate globally, making treatment critically challenging. To combat the prevailing situation, it is crucial to explore new anti-TB drugs with a novel mechanism of action and high efficacy. The Mycobacterium tuberculosis (M.tb)DciA is an essential protein involved in bacterial replication and regulates its growth. DciA interacts with DNA and provides critical help in binding other replication machinery proteins to the DNA. Moreover, the lack of any structural homology of M.tb DciA with human proteins makes it an appropriate target for drug development. In this study, FDA-approved drugs were virtually screened against M.tb DciA to identify potential inhibitors. Four drugs namely Lanreotide, Risedronate, Triflusal, and Zoledronic acid showed higher molecular docking scores. Further, molecular dynamics simulations analysis of DciA-drugs complexes reported stable interaction, more compactness, and reduced atomic motion. The anti-TB activity of drugs was further evaluated under in vitro and ex vivo conditions where Triflusal was observed to have the best possible activity with the MIC of 25 µg/ml. Our findings present novel DciA inhibitors and anti-TB activity of Triflusal. Further investigations on the use of Triflusal may lead to the discovery of a new anti-TB drug.

Purified cellobiose dehydrogenase of Termitomyces sp. OE147 fuels cellulose degradation resulting in the release of reducing sugars.

Termitomyces sp. OE 147 is one of the active cellulose degraders in the ecosphere and produces large amount of cellobiose dehydrogenase (CDH) and ß-glucosidases when cultivated on cellulose. In order to investigate its effect on cellulose, a highly purified preparation of CDH was obtained from the culture supernatant of the fungus cultivated on cellulose. A combination of ultrafiltration, ion-exchange and gel-filtration chromatography was used to purify CDH by ∼172-fold to a high specific activity of ∼324 U/mg protein on lactose which was used for routine measurement of enzyme activity. The enzyme displayed a pH optimum of 5.0 and stability between pH 5.0 and 8.0 with maximum catalytic efficiency (kcat/Km) of 397 mM-1 s-1 on cellobiose. Incubation of microcrystalline cellulose with the purified CDH led to production of reducing sugars which was accelerated by the addition of FeCl3 during the early stages of incubation. A mass spectrometric analysis revealed fragmentation products of cellulose which were concluded to be cellodextrins, sugars, and corresponding aldonic acids suggesting that CDH can release reducing sugars in the absence of externally added lytic polysaccharide monooxygenases. Polymerized products of glucose were also detected at low intensity.

Author Correction: Artificial Intelligence and Machine learning based prediction of resistant and susceptible mutations in Mycobacterium tuberculosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Artificial Intelligence and Machine learning based prediction of resistant and susceptible mutations in Mycobacterium tuberculosis.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M.tb), causes highest number of deaths globally for any bacterial disease necessitating novel diagnosis and treatment strategies. High-throughput sequencing methods generate a large amount of data which could be exploited in determining multi-drug resistant (MDR-TB) associated mutations. The present work is a computational framework that uses artificial intelligence (AI) based machine learning (ML) approaches for predicting resistance in the genes rpoB, inhA, katG, pncA, gyrA and gyrB for the drugs rifampicin, isoniazid, pyrazinamide and fluoroquinolones. The single nucleotide variations were represented by several sequence and structural features that indicate the influence of mutations on the target protein coded by each gene. We used ML algorithms - naïve bayes, k nearest neighbor, support vector machine, and artificial neural network, to build the prediction models. The classification models had an average accuracy of 85% across all examined genes and were evaluated on an external unseen dataset to demonstrate their application. Further, molecular docking and molecular dynamics simulations were performed for wild type and predicted resistance causing mutant protein and anti-TB drug complexes to study their impact on the conformation of proteins to confirm the observed phenotype.

Evaluation of cellobiose dehydrogenase and laccase containing culture fluids of Termitomyces sp. OE147 for degradation of Reactive blue 21.

This study evaluates culture filtrate, rich in cellobiose dehydrogenase and laccases, of Termitomyces sp. OE 147, in decolouration and degradation of Reactive blue (RB) 21. About 35% decolouration was achieved at low volumes of the culture supernatant without addition of external redox mediators. An optimized dye to culture fluid ratio (75 ppm: 0.1 ml) at a pH of 4-5 resulted in removal of colour by 60%. The degradation products of RB21 were analysed by Electron Spray Ionization-Mass Spectrometry and several small molecules (of m/z 106-199) were detected. These were concluded to be o-Xylene, 2,3-Dihydro-1H-isoindole, Isoindole-1,3-dione, 2,Benzenesulfonyl-ethanol, (4-Hydroxy-phenyl)-sulfamic acid, 2,3-Dihydro-1H-isoindole-5-sulfonic acid and proposed to result from joint action of cellobiose dehydrogenase, laccase, peroxidases and unidentified oxidoreductases present in the culture fluids. Based on the products formed and the known reactions of these enzymes, a degradation pathway was proposed for RB21. The culture fluid was also effective in decolouration (by about 50%) and detoxification (by ∼25%) of the combined effluent collected from a local mill indicating a treatment process that bypasses use of H2O2 and toxic mediators.

Differential production of lignocellulolytic enzymes by a white rot fungus Termitomyces sp. OE147 on cellulose and lactose.

White-rot fungi are the only organisms known to degrade all basic wood polymers using different strategies of employing a variety of hydrolytic and oxidative enzymes. A comparative secretome analysis of Termitomyces sp. OE147 cultivated on cellulose and lactose was carried out by two-dimensional gel electrophoresis followed by MALDI-TOF/TOF-MS analysis to identify the enzymes coordinately expressed on cellulose. A total of 29 proteins, belonging to CAZy hydrolases (11), CAZy oxidoreductases (13) and some 'other' (5) proteins were identified. Among the CAZy hydrolases, a distinct repertoire of cellulolytic and hemicellulolytic enzymes were produced while among the CAZy oxidoreductases, cellobiose dehydrogenase and laccase were the predominant enzymes along with H2O2 dependent peroxidases. This coordinated expression indicated a unique and integrated system for degradation of not only crystalline cellulose but also other components of lignocellulolytic substrates, namely lignin and xylan. Activities of the identified proteins were confirmed by plate assays and activity measurements. Many of the enzyme activities were also correlated with reduction in the crystallinity index of cellulose. Based on the enhanced production of CDH, ß-glucosidases and several oxidoreductases, a more prominent role of these enzymes is indicated in this fungus in cellulose breakdown.

Production of cellobiose dehydrogenase from a newly isolated white rot fungus Termitomyces sp. OE147.

Class I cellobiose dehydrogenases (CDHs) are extracellular hemoflavo enzymes produced at low levels by the Basidiomycetes (white rot fungi). In presence of suitable electron acceptors, e.g., cytochrome c, 2,6-dichlorophenol-indophenol, or metal ions, it oxidizes cellobiose to cellobionolactone. A stringent requirement for disaccharides makes CDH also useful for conversion of lactose to lactobionic acid, an important ingredient in pharma and detergent industry. In this work, class I CDH was produced using a newly identified white rot fungus Termitomyces sp. OE147. Four media were evaluated for CDH production, and maximum enzyme activity of 0.92 international unit (IU)/ml was obtained on Ludwig medium under submerged conditions. Statistical optimization of N source, which had significant effect on CDH production, using Box-Behnken design followed by optimization of inoculum size and age resulted in an increase in activity to 2.9 IU/ml and a productivity of ~25 IU/l/h. The nearly purified CDH exhibited high activity of 26.4 IU/mg protein on lactose indicating this enzyme to be useful for lactobionic acid synthesis. Some of the internal peptide sequences bore 100 % homology to the CDH produced in Myceliophthora thermophila. The fungal isolate was amenable to scale up, and an overall productivity of ~18 IU/l/h was obtained at 14-l level.